The present invention relates generally to certain proteins and to methods of improving the biological activity of certain proteins, and more specifically, chemokines.
All the members of the intercrine or chemokine family are basic heparin-binding polypeptides which have four cysteine residues which form two disulfide bridges. All these proteins which have been functionally characterized appear to be involved in proinflammatory and/or restorative functions. As such, these molecules are anticipated to have therapeutic potential in bone marrow transplantation and the treatment of infections, cancer, myelopoietic dysfunction, graft versus host disease, and autoimmune diseases.
The chemokine family can be divided into two subfamilies depending upon their amino acid sequence and chromosomal location. The members of the xcex1 subfamily are termed the xe2x80x9cC-X-Cxe2x80x9d subfamily because the first two cysteines are separated by only one amino acid. The human genes in this subfamily include IL-8, GRO/MGSA, and IP-10; murine counterparts include KC and macrophage inflammatory protein 2 (MIP-2). In the chemokine xcex2 subfamily, the first two cysteines are in an adjacent position (the xe2x80x9cC-Cxe2x80x9d subfamily). This subfamily includes human MCAF, LD-78, ACT-2, and RANTES. The murine counterparts are JE, TCA-3, MIP-1xcex1, and MIP-1xcex2 [J. J. Oppenheim et al, Annu. Rev. Immunol., 9:617-648 (1991)].
The murine KC gene product [Oquendo et al, J. Biol. Chem., 264:4233 (1989)] is induced by platelet-derived growth factor (PDGF) and this is thought to be the murine homolog of the human MGSA/groxcex1 gene (63.0% amino acid identity to mMIP-2). KC has been expressed in COS-1 cells to show that it encodes a secreted protein [Oquendo, cited above].
Two forms of MIP have been found in cultures of macrophage tumor cells from the mouse: MIP-1 and MIP-2. Murine MIP-2 (mMIP-2) is an inducible protein whose cDNA also has been cloned and sequenced [International Patent Application, Publication No. WO 90/02762 (Mar. 22, 1990)]. Murine MIP-2 has been shown to have potent chemotactic activity for human polymorphonuclear leukocytes (PMN), and to induce PMN degranulation of lysozyme but not of xcex2-glucuronidase [Wolpe et al, J. Exp. Med., 167:570 (1987)]. Further, mMIP-2 has been shown to have myelopoietic enhancing activities for CFU-GM [Broxmeyer et al, J. Exp. Med., 170:1583 (1989)]. The human counterpart of this factor was found to consist of two species, MIP-2xcex1 and MIP-2xcex2, also termed gro-xcex2 and gro-xcex3, respectively.
The cDNA and amino acid sequences of human gro-xcex2 are provided in International Patent Application, Publication No. WO 92/00327 (Jan. 9, 1992); the cDNA and amino acid sequences of human gro-xcex3 are provided in International Patent Application, Publication No. WO 92/00326 (Jan. 9, 1992). Each of these sequences were predicted to encode a 73 amino acid mature protein.
MGSA or gro-xcex1 [Richmond et al, EMBO J., 7:2025 (1988)] is an autocrine growth factor with potent mitogenic activity secreted by human melanoma cells and is the product of the human gro gene [Anisowicz et al, Proc. Natl. Acad. Sci., 84:7188 (1987)].
There remains a need in the art for methods of enhancing the bioactivity of these mature proteins to enable their efficient use as therapeutic or pharmaceutical products, and to minimize the amounts of the proteins necessary to produce a therapeutic effect, thereby lowering toxicity.
In one aspect, the present invention provides a modified chemokine, which includes KC protein, human gro xcex1, gro-xcex2, and gro-xcex3, which modified protein is characterized by truncation of between about 2 to about 8 amino acids at the amino terminus of the mature protein and by at least a log higher biological activity than the mature protein.
In another aspect, the present invention provides a modified chemokine which is characterized by truncation of between about 2 to about 10 amino acids at the carboxy terminus of the mature protein and by at least a log higher biological activity than the mature protein.
In still another aspect, the present invention provides a multimeric protein which comprises an association of two or more modified proteins of this invention. These multimers preferably contain multiple copies of the same modified protein, e.g., a dimer of truncated KC protein. However, multimeric forms of two or more different modified proteins of this invention are also included in this invention. Multimeric forms of a modified protein of this invention and another known mature protein are also encompassed by this invention.
In a further aspect, the present invention provides a method of enhancing the biological activity of chemokines by modifying and/or truncating the proteins as described above.
In yet another aspect, the present invention provides pharmaceutical and diagnostic compositions containing the modified and multimeric proteins of the invention, as well as methods for administering same in therapeutic treatments.
In a further aspect, the present invention provides antibodies characterized by the ability to selectively bind the modified chemokines of the invention.
In still another aspect, the present invention provides a method of monitoring the effect of a selected hematopoiesis stimulating agent upon hematopoietic synergistic factor (HSF) in vivo through use of an antibody of the invention.
In yet another aspect, the present invention provides a method of inducing HSF in vivo by administering (pGlu-Glu-Asp)2-Sub-(Lys)2 [SEQ ID NO: 5].
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.